Bestrophin and bestrophin homologous proteins involved in the regulation of energy homeostasis

ABSTRACT

The present invention discloses Bestrophin homologous proteins regulating the energy homeostasis and the metabolism of triglycerides, and polynucleotides, which identify and encode the proteins disclosed in this invention. The invention also relates to the use of these sequences in the diagnosis, study, prevention, and treatment of diseases and disorders, for example, but not limited to, metabolic diseases such as obesity as well as related disorders such as eating disorder cachexia, diabetes mellitus, hypertension, coronary heart disease, hypercholesterolemia, osteoarthritis, gallstones, cancers of the reproductive organs, and sleep apnea.

This invention relates to the use of nucleic acid and amino acidsequences of Bestrophin and Bestrophin homologous proteins (for example,VMD2, VMD2-like protein 1, VMD2-like protein 2, or VMD2-like protein 3),and to the use of these sequences and effectors thereof in thediagnosis, study, prevention, and treatment of diseases and disorders,for examples but not limited to, metabolic diseases such as obesity,body-weight regulation, thermogenesis as well as related disorders suchas eating disorder, cachexia, diabetes mellitus, hypertension, coronaryheart disease, hypercholesterolemia, osteoarthritis, gallstones, cancersof the reproductive organs, and sleep apnea.

Obesity is one of the most prevalent metabolic disorders in the world.It is a still poorly understood human disease that becomes more and morerelevant for western society. Obesity is defined as an excess of bodyfat, frequently resulting in a significant impairment of health. Besidessevere risks of illness such as diabetes, hypertension and heartdisease, individuals suffering from obesity are often isolated socially.Obesity is influenced by genetic, metabolic, biochemical, psychological,and behavioral factors. As such, it is a complex disorder that must beaddressed on several fronts to achieve lasting positive clinicaloutcome. Obese individuals are prone to ailments including: diabetesmellitus, hypertension, coronary heart disease, hypercholesterolemia,osteoarthritis, gallstones, cancers of the reproductive organs, andsleep apnea.

Obesity is not to be considered as a single disorder but a heterogeneousgroup of conditions with (potential) multiple causes. For example,obesity is also characterized by elevated fasting plasma insulin and anexaggerated insulin response to oral glucose intake (Koltermann, J.Clin. Invest 65, 1980, 1272-1284) and a clear involvement of obesity intype 2 diabetes mellitus can be confirmed (Kopelman, Nature 404, 2000,635-643).

Even if several candidate genes have been described which are supposedto influence the homeostatic system(s) that regulate body mass/weight,like leptin, VCPI, VCPL, or the peroxisome proliferator-activatedreceptor (PPAR)-gamma co-activator, the distinct molecular mechanismsand/or molecules influencing obesity or body weight/body massregulations are not known.

Therefore, the technical problem underlying the present invention was toprovide for means and methods for modulating (pathological) metabolicconditions influencing thermogenesis, body-weight regulation and/orenergy homeostatic circuits. The solution to said technical problem isachieved by providing the embodiments characterized in the claims.

Accordingly, the present invention relates to a nucleic acid of theBestrophin gene family, particularly the human Bestrophin genes (forexample, VMD2, VMD2-like protein 1, VMD2-like protein 2, or VMD2-likeprotein 3) having novel functions in body-weight regulation, energyhomeostasis, metabolism, and obesity as well as related disorders suchas eating disorder, cachexia, diabetes mellitus, hypertension, coronaryheart disease, hypercholesterolemia, osteoarthritis, gallstones, cancersof the reproductive organs, and sleep apnea. The nucleic acid, theprotein coding therefore and an antibody, aptamer or another receptorrecognizing the nucleic acid or the protein may be used for diagnosticor therapeutic purposes or as a target for the development of novelagents.

One gene of the human Bestrophin family (also referred to as VMD2) isexpressed in several tissues including the retinal pigment epithelium(RPE) where it is localized at the epithelial basolateral plasmamembrane (Petrukhin et al., 1998, Nat Genet 19: 241-247; Marmorstein etal., 2000, Proc Natl Acad Sci USA, 97(23):12758-12763). Heterozygousmutations of the Bestrophin (VDM2) gene are associated with Best maculardystrophy (BMD). Best macular dystrophy is a dominantly inherited, earlyonset disease of macular degeneration that may develop subretinalneovascularisation similar to the wet type of age-related maculardegeneration. Macular degeneration is a leading cause of blindness thataffects the aged population. Best vitelliform macular dystrophy (VDMZ)is a macular degeneration characterized by the deposition oflipofuscin-like material within and below the RPE and is associated withdegeneration of the RPE and overlying photoreceptors (Allikmets, 1999,Hum Genet 104(6):449-453). Bestrophin is the protein identified as beingresponsible for VDM2 and possibly other forms of maculopathy withphenotypic characteristics similar to Best disease.

Structural computer analysis of the Bestrophin gene sequence suggestthat the encoded peptide is a transmembrane protein that defines a newfamily of anion, in particular chloride channels (ion exchangers)(Marmorstein A D. et al. 2000, supra; Gomez A. et al. 2001, DNA Seq12(5-6):431-435; Sun et al., 2002, PNAS 99(6), 4008-4013). Bestrophinsfrom different species can form oligomeric chloride (anion, nitrate,bicarbonate) channels responsible for the calcium sensitive chlorideconductance in RPE cells. Loss of chloride conductance can also impairthe co-transport of nutrients and other essential molecules in RPE whichleads to the observed degeneration of RPE, including accumulation oflipofuscin-like material within and below the RPE (Sun H. et al., 2002,supra).

It has recently been shown that Bestrophin interacts physically andfunctionally with Protein Phosphatase 2A (PP2A) (Marmorstein, 2002, J.Biol. Chem. 277(34), 30591-30597). The beta catalytic subunit of PP2A(PP2Ac) and the structural scaffold subunit of PP2A wereimmunoprecipitated together with Bestrophin. Bestrophin isphosphorylated in RPE-J cells that is sensitive to the proteinphosphatase inhibitor okadaic acid. It was shown that PP2Adephosphorylates Bestrophin in vitro suggesting the regulation of theBestrophin anion channel activity through PP2A. Therefore, Bestrophin isa member of the signal transduction-pathway that modulates the lightpeak in the eye (Marmorstein et al. 2002, supra).

Recently, Bestrophin overexpression was shown to directly influence thechloride conductance of HEK293 cells, which led the authors conclude toassign chloride channel activity to bestrophin and thereby to suggestVMD as channelopathy (Sun et al., 2002, supra). Furthermore, thephysical interaction between bestrophin and a protein phosphatase, PP2A,suggests that phosphorylation or dephosphorylation of bestrophin may actas the on/off switch for the light peak or modulate its amplitude ortiming (Marmorstein, 2002, supra).

Although a clear function of Bestrophins in Best macular dystrophy hasbeen shown, no function in the regulation of energy homeostasis has beendescribed up to date. Surprisingly, we found that Bestrophins areinvolved in the regulation of energy homeostasis. A genetic screen wasused to identify that mutation of a Bestrophin homologous gene causesobesity, reflected by a significant increase of triglyceride content,the major energy storage substance. In this invention we demonstratethat the correct gene dose of Bestrophin and Bestrophin homologousproteins is essential for maintenance of energy homeostasis. Inaddition, we found that the expression of Bestrophins is high in thehypothalamus and other brain areas suggesting roles in the regulation ofthe metabolism. We could clearly see that VDM2-like protein 3 and VDM2are ubiquitously expressed with high expression in white and brownadipose tissue. We could also see that the expression of VMD2 likeprotein 3 is downregulated in genetically obese mice and in mice underhigh fat diet, whereas expression of VMD2 is upregulated in those obesemice. Further, VMD2 expression is upregulated in human preadipocytes.

Based on these results, it can be concluded that polynucleotidesencoding proteins with homologies to Bestrophins (for example, VMD2,VMD2-like protein 1, VMD2-like protein 2, or VMD2-like protein 3)present the opportunity to investigate diseases and disorders asdescribed above. Thus, new compositions useful in diagnosis, treatment,and prognosis of metabolic diseases and disorders and related diseasesare provided.

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meanings as commonly understood by one of ordinary skill in theart to which this invention belongs. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure.

The present invention discloses that Bestrophin homologous proteins (forexample, VMD2, VMD2-like protein 1, VMD2-like protein 2, or VMD2-likeprotein 3) encoded by nucleic acids of the Bestrophin gene family areregulating the energy homeostasis and fat metabolism, especially themetabolism and storage of triglycerides. The invention also relates tovectors, host cells, receptors or effectors of the proteins or nucleicacids such as antibodies, aptamers, peptides, antisense molecules,ribozymes, RNAi molecules or low molecular weight inhibitors oractivators and recombinant methods for producing the polypeptides andpolynucleotides of the invention. The invention also relates to the useof these molecules in the diagnosis, study, prevention, and treatment ofdiseases and disorders as described above.

The term “GenBank Accession number” relates to NCBI GenBank databaseentries (Benson et al, Nucleic Acids Res. 28, 2000, 15-18).).

Bestrophin homologous proteins and nucleic acid molecules codingtherefore are obtainable from insect or vertebrate species, e.g. mammalsor birds. Particularly preferred are human Bestrophin homologous nucleicacids, particularly nucleic acids encoding a

-   (i) a human Bestrophin protein on chromosome 11 (or also refered to    as human vitelliform macular dystrophy, VMD2; Genbank Accession No.    NM_(—)004183 and No. NP_(—)004174; Swiss Prot. Accession    Number_(—)076090; formerly Genbank Accession No. XM_(—)043405; see    FIGS. 4A and 4B), or-   (ii) a Bestrophin homologous protein on human chromosome 12 (genomic    sequence Genbank Accession No. AC016153, identical to human    VMD2-like protein 3, vitelliform macular dystrophy 2-like protein 3;    Genbank Accession Number AF440758_(—)1, see FIGS. 4E and 4F),-   (iii) a Bestrophin homologous protein on human chromosome 1 (genomic    sequence Genbank Accession No. AL592166, identical to encoding a    human VMD2-like protein 2, vitelliform macular dystrophy 2-like    protein 2; Genbank Accession Number AF440757_(—)1, see FIGS. 4G and    4H), or-   (iv) a Bestrophin homologous protein on human chromosome 19    (assembled from cDNA Genbank Accession No. NM_(—)017682 and genomic    sequence AC018761, similar to human VMD2-like protein 1, vitelliform    macular dystrophy 2-like protein 1 (Genbank Accession Number    AF440756_(—)1) which has an additional 43 amino acids at the amino    terminus, see FIGS. 4C and 4D or FIGS. 41 and 4J).

The invention particularly relates to a nucleic acid molecule encoding apolypeptide contributing to regulating the energy homeostasis and themetabolism of triglycerides or a portion thereof, wherein said nucleicacid molecule comprises

-   -   (a) a Bestrophin nucleotide sequence as shown in FIG. 4 or the        complementary strand thereof,    -   (b) a nucleotide sequence which hybridizes under stringent        conditions to a nucleic acid molecule encoding a Bestrophin        amino acid sequence as shown in FIG. 4 or the complementary        strand thereof,    -   (c) a nucleotide sequence corresponding to the sequences of (a)        or (b) within the degeneration of the genetic code, (d) a        nucleotide sequence which encodes a polypeptide which is at        least 85%, preferably at least 90%, more preferably at least        95%, more preferably at least 98% and up to 99.6% identical to        an amino acid sequence as shown in FIG. 4,    -   (e) a nucleotide sequence which differs from the nucleic acid        molecule of (a) to (d) by mutation and wherein said mutation        causes an alteration, deletion, duplication or premature stop in        the encoded polypeptide, or    -   (f) a partial nucleotide sequence of any of the sequences of (a)        to (e) having a length of at least 15 bases, preferably at least        20 bases, more preferably at least 25 bases and most preferably        at least 50 bases.

The invention is based on the result that Bestrophin homologous proteins(for example, VMD2, VMD2-like protein 1, VMD2-like protein 2, orVMD2-like protein 3; herein also referred to as Bestrophin orBestrophins) and the polynucleotides encoding these, are involved in theregulation of triglyceride storage and therefore energy homeostasis.Mostly preferred are nucleic acids encoding VMD2 or VMD2-like protein 3and the corresponding proteins and effectors thereof.

The invention describes the use of these compositions for the diagnosis,study, prevention, or treatment of diseases and disorders relatedthereto, including metabolic diseases such as obesity as well as relateddisorders such as eating disorder, cachexia, diabetes mellitus,hypertension, coronary heart disease, hypercholesterolemia,osteoarthritis, gallstones, cancers of the reproductive organs, andsleep apnea.

To find genes with novel functions in energy homeostasis, metabolism,and obesity, a functional genetic screen was performed with the modelorganism Drosophila melanogaster (Meigen). One resource for screeningwas a Drosophila melanogaster stock collection of EP-lines. The P-vectorof this collection has Gal4-UAS-binding sites fused to a basal promoterthat can transcribe adjacent genomic Drosophila sequences upon bindingof Gal4 to UAS-sites. This enables the EP-line collection foroverexpression of endogenous flanking gene sequences. In addition,without activation of the UAS-sites, integration of the EP-element intothe gene is likely to cause a reduction of gene activity, and allowsdetermining its function by evaluating the loss-of-function phenotype.

Triglycerides are the most efficient storage for energy in cells. Obesepeople mainly show an significant increase in the content oftriglycerides. In order to isolate genes with a function in energyhomeostasis, several thousand EP-lines were tested for theirtriglyceride content after a prolonged feeding period. Lines withsignificantly changed triglyceride content were selected as positivecandidates for further analysis as, for example, but not for limitingthe scope of the invention, is described below in the examples section.

The result of the triglyceride content analysis is shown in FIG. 1. Wefound that homozygous HD-EP(3)32517 and HD-EP(3)36237 flies have ahigher triglyceride content than the controls (average triglyceridelevels). Therefore, the very likely loss of a gene activity in the genelocus 85F13-85F14 (estimated chromosomal localisation where theEP-vector of HD-EP(3)32517 and HD-EP(3)36237 flies is integrated) isresponsible for changes in the metabolism of the energy storagetriglycerides, therefore representing in both cases an obese fly model.

The increase of triglyceride content due to the loss of a gene functionsuggests gene activities in energy homeostasis in a dose dependentmanner that controls the amount of energy stored as triglycerides.

Nucleic acids encoding the Bestrophin protein of the present inventionwere identified using a plasmid-rescue technique. Genomic DNA sequenceswere isolated that are localised directly 5′ or 3′ to the EP(3)32517 andHD-EP(3)36237 integrations. Using those isolated genomic sequencespublic databases like Berkeley Drosophila Genome Project (GadFly) werescreened thereby confirming the integration side of EP(3)32517 andHD-EP(3)36237 within a 5′ exon or enhancer/promoter region of theBestrophin homologous gene (FIG. 2). FIG. 2 shows the molecularorganisation of this locus. Genomic DNA sequence is represented by theassembly as a black dotted line in the middle that includes theintegration site of EP(3)32517 and HD-EP(3)36237. Numbers represent thecoordinates of the genomic DNA. Grey bars on the two cDNA-linesrepresent the predicted genes (GadFly & Magpie), and grey symbols on theP-Elements-line the EP-vector integration sites. Predicted exons of geneCG6264 are shown as dark grey bars and predicted introns as light greybars.

Bestrophin encodes for a gene that is predicted by GadFly sequenceanalysis programs (GadFly Accession Number CG6264). No functional datadescribed the regulation of obesity and metabolic diseases are availablein the prior art for the genes and proteins shown in FIGS. 3, 4, and 5,referred to as Bestrophin or Bestrophin homologues in the presentinvention.

The present invention further relates to a polypeptide encoded by thenucleic acid as described above. Preferably the polypeptide comprisesthe amino acid sequence of Bestrophin. A comparison (Clustal X 1.8 orClustaW 1.82) between the Bestrophin proteins of different species(human, mouse, and Drosophila) was conducted and is shown in FIGS. 3 and5.

Using TMHMM protein analysis tools (A. Krogh, et al., Journal ofMolecular Biology, 305(3):567-580, 2001.), it was found, for example,that the Bestrophin protein of the invention has at least fourcharacteristic protein motifs (for example, transmembrane domains).These motifs are found throughout the whole Bestrophin famliy. InterProanalysis (Apweiler et al., Nucleic Acids Research 29:37-40, 2001) of theDrosophila gene (CG6264) indicated the presence of a worm-family-8domain (Sonnhammer and Durbin, Genomics 46:200-216, 1997) typical forall Bestrophin homologous proteins. Based upon homology, Bestrophinproteins of the invention and each homologous protein or peptide mayshare at least some activity.

As shown in FIG. 6, VDM2-like protein 3 shows clear expression inadipose tissues. Under high fat diet, VDM2-like protein 3 isdown-regulated in adipose tissues (WAT), suggesting that the protein isregulating the adipogenesis, possibly as inhibitor of this process. Theexpression of three Bestrophiris (VMD2, VMD2-like protein 1, VMD2-likeprotein 3) was observed in adipose tissues as well as in thehypothalamus and to a lesser degree in other brain areas. Thus,Bestrophins show a clear tissue specific expression suggesting distinctroles in the metabolism (see Examples 4 and 5 and FIGS. 6, 7 and 8 andFIGS. 9 and 10 respectively).

The invention also encompasses polynucleotides that encode Bestrophinand homologous proteins. Accordingly, any nucleic acid sequence, whichencodes the amino acid sequences of Bestrophin, can be used to generaterecombinant molecules that express Bestrophin. In a particularembodiment, the invention encompasses polynucleotides selected fromhuman Bestrophin nucleic acids as described above or fragments orvariants thereof. It will be appreciated by those skilled in the artthat as a result of the degeneracy of the genetic code, a multitude ofnucleotide sequences encoding Bestrophins, some bearing minimal homologyto the nucleotide sequences of any known and naturally occurring gene,may be produced. Thus, the invention contemplates each and everypossible variation of nucleotide sequence that could be made byselecting combinations based on possible codon choices. Thesecombinations are made in accordance with the standard triplet geneticcode as applied to the nucleotide sequences of naturally occurringBestrophins, and all such variations are to be considered as beingspecifically disclosed. Although nucleotide sequences which encodeBestrophins and their variants are preferably capable of hybridising tothe naturally occurring nucleotide sequences of Bestrophins (for examplethe sequences encoding VDM2, VDM2-like protein 1, VDM2-like protein 2,or VDM2-like protein 3) under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequences ortheir derivatives possessing a substantially different codon usage.Codons may be selected to increase the rate at which expression of thepeptide occurs in a particular prokaryotic or eukaryotic host inaccordance with the frequency with which particular codons are utilisedby the host. Other reasons for substantially altering the nucleotidesequence encoding Bestrophins and its derivatives without altering theencoded amino acid sequences include the production of RNA transcriptshaving more desirable properties, such as a greater half-life, thantranscripts produced from the naturally occurring sequences. Theinvention also encompasses production of DNA sequences or portionsthereof and its derivatives, entirely by synthetic chemistry. Afterproduction, the synthetic sequence may be inserted into any of the manyavailable expression vectors and cell systems using reagents that arewell known in the art at the time of the filing of this application.Moreover, synthetic chemistry may be used to introduce mutations into asequence encoding Bestrophin or any portion thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed nucleotide sequences, undervarious conditions of stringency. Hybridization conditions are based onthe melting temperature (Tm) of the nucleic acid binding complex orprobe, as taught in Wahl, G. M. and S. L. Berger (1987: Methods Enzymol.152:399-407) and Kimmel, A. R. (1987; Methods Enzymol. 152:507-511), andmay be used at a defined stringency. Preferably, hybridization understringent conditions means that after washing for 1 h with 1×SSC and0.1% SDS at 50° C., preferably at 55° C., more preferably at 62° C. andmost preferably at 68° C., particularly for 1 h in 0.2×SSC and 0.1% SDSat 50° C., preferably at 55° C., more preferably at 62° C. and mostpreferably at 68° C., a positive hybridization signal is observed.Altered nucleic acid sequences encoding Bestrophin which are encompassedby the invention include deletions, insertions, or substitutions ofdifferent nucleotides resulting in a polynucleotide that encodes thesame or a functionally equivalent Bestrophin.

The encoded proteins may also contain deletions, insertions, orsubstitutions of amino acid residues, which produce a silent change andresult in a functionally equivalent Bestrophin (for example, VDM2,VDM2-like protein 1, VDM2-like protein 2, or VDM2-like protein 3).Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological activity of Bestrophin is retained. For example,negatively charged amino acids may include aspartic acid and glutamicacid; positively charged amino acids may include lysine and arginine;and amino acids with uncharged polar head groups having similarhydrophilicity values may include leucine, isoleucine, and valine;glycine and alanine; asparagine and glutamine; serine and threonine;phenylalanine and tyrosine.

Also included within the scope of the present invention are alleles ofthe genes encoding Bestrophins (for example, VDM2, VDM2-like protein 1,VDM2-like protein 2, or VDM2-like protein 3). As used herein, an“allele” or “allelic sequence” is an alternative form of the gene, whichmay result from at least one mutation in the nucleic acid sequence.Alleles may result in altered mRNAs or polypeptides whose structures orfunction may or may not be altered. Any given gene may have none, one,or many allelic forms. Common mutational changes, which give rise toalleles, are generally ascribed to natural deletions, additions, orsubstitutions of nucleotides. Each of these types of changes may occuralone, or in combination with the others, one or more times in a givensequence. These changes may be determined by DNA sequencing methodswhich are well known and generally available in the art.

The nucleic acid sequences encoding Bestrophin (for example, VDM2,VDM2-like protein 1, VDM2-like protein 2, or VDM2-like protein 3) may beextended utilising a partial nucleotide sequence and employing variousmethods known in the art to detect upstream sequences such as promotersand/or regulatory elements. For example, one method which may beemployed, “restriction-site” PCR, uses universal primers to retrieveunknown sequence adjacent to a known locus (Sarkar, G. (1993) PCRMethods Applic. 2:318-322). Inverse PCR may also be used to amplify orextend sequences using divergent primers based on a known region(Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186). Another methodwhich may be used is capture PCR which involves PCR amplification of DNAfragments adjacent to a known sequence in human and yeast artificialchromosome DNA (Lagerstrom, M. et al. (PCR Methods Applic. 1:111-119).Another method which may be used to retrieve unknown sequences is thatof Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PROMOTERFINDERlibraries to walk in genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences, which contain the 5′ regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into the 5′ and 3′non-transcribed regulatory regions. Capillary electrophoresis systems,which are commercially available, may be used to analyse the size orconfirm the nucleotide sequence of sequencing or PCR products.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode Bestrophin, or fusion proteins orfunctional equivalents thereof, may be used in recombinant DNA moleculesto direct expression of Bestrophin in appropriate host cells. Due to theinherent degeneracy of the genetic code, other DNA sequences, whichencode substantially the same, or a functionally equivalent amino acidsequence may be produced and these sequences may be used to clone andexpress Bestrophin. As will be understood by those of skill in the art,it may be advantageous to produce Bestrophin encoding nucleotidesequences possessing non-naturally occurring codons. For example, codonspreferred by a particular prokaryotic or eukaryotic host can be selectedto increase the rate of protein expression or to produce a recombinantRNA transcript having desirable properties, such as a half-life which islonger than that of a transcript generated from the naturally occurringsequence. The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterBestrophin encoding sequences for a variety of reasons, including butnot limited to, alterations which modify the cloning, processing, and/orexpression of the gene product. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides maybe used to engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, or introduce mutations, and so forth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding Bestrophin (for example,VDM2, VDM2-like protein 1, VDM2-like protein 2, or VDM2-like protein 3)may be ligated to a heterologous sequence to encode a fusion protein.For example, to screen peptide libraries for modulators, e.g. inhibitorsof Bestrophin activities, it may be useful to use chimeric Bestrophinproteins that can be recognised by a commercially available antibodies.A fusion protein may also be engineered to contain a cleavage sitelocated between the Bestrophin encoding sequence and the heterologousprotein sequences, so that Bestrophin may be cleaved and purified awayfrom the heterologous moiety. In another embodiment, sequences encodingBestrophin may be synthesised, in whole or in part, using chemicalmethods well known in the art (see Caruthers, M. H. et al. (1980) Nucl.Acids Res. Symp. Ser. 7:215-223, Horn, T. et al. (1980) Nucl. Acids Res.Symp. Ser. 7:225-232). Alternatively, the proteins themselves may beproduced using chemical methods to synthesise the amino acid sequence ofBestrophin, or a portion thereof. For example, peptide synthesis can beperformed using various solid-phase techniques (Roberge, J. Y. et al.(1995) Science 269:202-204) and automated synthesis may be achieved, forexample, using the ABI 431A peptide synthesiser (Perkin Elmer). Thenewly synthesised peptide may be substantially purified by preparativehigh performance liquid chromatography (e.g., Creighton, T. (1983)Proteins, Structures and Molecular Principles, WH Freeman and Co., NewYork, N.Y.). The composition of the synthetic peptides may be confirmedby amino acid analysis or sequencing (e.g., the Edman degradationprocedure; Creighton, supra). Additionally, the amino acid sequences ofBestrophin, or any part thereof, may be altered during direct synthesisand/or combined using chemical methods with sequences from otherproteins, or any part thereof, to produce a variant polypeptide.

In order to express a biologically active Bestrophin (for example, VDM2,VDM2-like protein 1, VDM2-like protein 2, or VDM2-like protein 3), thenucleotide sequences encoding Bestrophin functional equivalents, may beinserted into appropriate expression vectors, i.e., a vector, whichcontains the necessary elements for the transcription and translation ofthe inserted coding sequence. Methods, which are well known to thoseskilled in the art, may be used to construct expression vectorscontaining sequences encoding Bestrophin and appropriate transcriptionaland translational control elements. These methods include in vitrorecombinant DNA techniques synthetic techniques, and in vivo geneticrecombination. Such techniques are described in Sambrook, J. et al.(1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press,Plainview, N.Y., and Ausubel, F. M. et al. (1989) Current Protocols inMolecular Biology, John Wiley & Sons, New York, N.Y.

A variety of expression vector/host systems may be utilised to containand express sequences encoding Bestrophin (for example, VDM2, VDM2-likeprotein 1, VDM2-like protein 2, or VDM2-like protein 3). These include,but are not limited to, micro-organisms such as bacteria transformedwith recombinant bacteriophage, plasmid, or cosmid DNA expressionvectors; yeast transformed with yeast expression vectors; insect cellsystems infected with virus expression vectors (e.g., baculovirus);plant cell systems transformed with virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or withbacterial expression vectors (e.g., Ti or PBR322 plasmids); or animalcell systems. The vectors may comprise “control elements” or “regulatorysequences”, i.e. non-translated regions including enhancers, promoters,5′ and 3′ untranslated regions which interact with host cellularproteins to carry out transcription and translation. Such elements mayvary in their strength and specificity. Depending on the vector systemand host utilised, any number of suitable transcription and translationelements, including constitutive and inducible promoters, may be used.For example, when cloning in bacterial systems, inducible promoters suchas the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene,LaJolla, Calif.) or PSPORT1 plasmid (Gibco BRL) and the like may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters and enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters and leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequences encodingBestrophin, vectors based on SV40 or EBV may be used with an appropriateselectable marker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for Bestrophin. For example, when largequantities of Bestrophin are needed for the induction of antibodies,vectors, which direct high level expression of fusion proteins that arereadily purified, may be used. Such vectors include, but are not limitedto, the multifunctional E. coli cloning and expression vectors such asthe BLUESCRIPT phagemid (Stratagene), pIN vectors (Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509); and the like. PGEXvectors (Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with Glutathione S-Transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will. In the yeast, Saccharomycescerevisiae, a number of vectors containing constitutive or induciblepromoters such as alpha factor, alcohol oxidase, and PGH may be used.For reviews, see Ausubel et al., (supra) and Grantet al. (1987) MethodsEnzymol. 153:5.16-544.

In cases where plant expression vectors are used, the expression ofsequences encoding Bestrophin may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J.3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter,J. et al. (1991) Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).

An insect system may also be used to express Bestrophin. For example, inone such system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae (Engelhard, E. K. et al.(1994) Proc. Nat. Acad. Sci. 91:3224-3227).

In mammalian host cells, a number of non-viral or viral expressionsystems may be utilised. In cases where an adenovirus is used as anexpression vector, sequences encoding Bestrophin may be ligated into anadenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a non-essential E1or E3 region of the viral genome may be used to obtain viable viruseswhich are capable of expressing Bestrophin in infected host cells(Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). Inaddition, transcription enhancers, such as the Rous sarcoma virus (RSV)enhancer, may be used to increase expression in mammalian host cells.

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, andWI38, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressBestrophin may be transformed using expression vectors which may containviral origins of replication and/or endogenous expression elements and aselectable marker gene on the same or on a separate vector. Followingthe introduction of the vector, cells may be allowed to grow for 1-2days in an enriched media before they are switched to selective media.The purpose of the selectable marker is to confer resistance toselection, and its presence allows growth and recovery of cells, whichsuccessfully express the introduced sequences. Resistant clones ofstably transformed cells may be proliferated using tissue culturetechniques appropriate to the cell type. Any number of selection systemsmay be used to recover transformed cell lines. These include, but arenot limited to, the herpes simplex virus thymidine kinase (Wigler, M. etal. (19.77) Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy,I. et al. (1980) Cell 22:817-23) genes, which can be employed in tk⁻ oraprt⁻ cells, respectively. Also, antimetabolite, antibiotic or herbicideresistance can be used as the basis for selection; for example, dhfrwhich confers resistance to methotrexate (Wigler, M. et al. (1980) Proc.Natl. Acad. Sci. 77:3567-70); npt, which confers resistance to theaminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al (1981) J.Mol. Biol. 150:1-14) and als or pat, which confer resistance tochlorsulfuron and phosphinotricin acetyltransferase, respectively(Murry, supra). Additional selectable genes have been described, forexample, trpB, which allows cells to utilise indole in place oftryptophan, or hisD, which allows cells to utilise histinol in place ofhistidine (Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad.Sci. 85:8047-51). Recently, the use of visible markers has gainedpopularity with such markers as anthocyanins, β-glucuronidase and itssubstrate GUS, and luciferase and its substrate luciferin, being widelyused not only to identify transformants, but also to quantify the amountof transient or stable protein expression attributable to a specificvector system (Rhodes, C. A. et al. (1995) Methods Mol. Biol.55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequences encoding Bestrophinare inserted within a marker gene sequence, recombinant cells containingsequences encoding Bestrophin can be identified by the absence of markergene function. Alternatively, a marker gene can be placed in tandem withsequences encoding Bestrophin under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well. Alternatively,host cells, which contain the nucleic acid sequences encoding Bestrophinand express Bestrophin, may be identified by a variety of proceduresknown to those of skill in the art. These procedures include, but arenot limited to, DNA-DNA, or DNA-RNA hybridisation and protein bioassayor immunoassay techniques which include membrane, solution, or chipbased technologies for the detection and/or quantification of nucleicacid or protein.

The presence of polynucleotide sequences encoding Bestrophin can bedetected by DNA-DNA or DNA-RNA hybridisation or amplification usingprimers, probes or portions or fragments of polynucleotides encodingBestrophin. Nucleic acid amplification based assays involve the use ofoligonucleotides or oligomers based on the sequences encoding Bestrophinto detect transformants containing DNA or RNA encoding Bestrophin. Asused herein “oligonucleotides” or “oligomers” refer to a nucleic acidsequence of at least about 10 nucleotides and as many as about 60nucleotides, preferably about 15 to 30 nucleotides, and more preferablyabout 20-25 nucleotides, which can be used as a probe or amplimer.

A variety of protocols for detecting and measuring the expression ofBestrophin, using either polyclonal or monoclonal antibodies specificfor the protein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilising monoclonal antibodies reactive to two non-interfering epitopeson Bestrophin is preferred, but a competitive binding assay may beemployed. These and other assays are described, among other places, inHampton, R. et al. (1990; Serological Methods, a Laboratory Manual, APSPress, St Paul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labelled hybridisation or PCR probesfor detecting sequences related to polynucleotides encoding Bestrophininclude oligo-labelling, nick translation, end-labelling or PCRamplification using a labelled nucleotide.

Alternatively, the sequences encoding Bestrophin (for example, VDM2,VDM2-like protein 1, VDM2-like protein 2, or VDM2-like protein 3), orany portion thereof may be cloned into a vector for the production of anmRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesise RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labellednucleotides. These procedures may be conducted using a variety ofcommercially available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.);Promega (Madison Wis.); and U.S. Biochemical Corp., (Cleveland, Ohio).

Suitable reporter molecules or labels, which may be used, includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, co-factors, inhibitors, magneticparticles, and the like.

Host cells transformed with nucleotide sequences encoding Bestrophin maybe cultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a recombinantcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeBestrophin may be designed to contain signal sequences, which directsecretion of Bestrophin through a prokaryotic or eukaryotic cellmembrane. Other recombinant constructs may be used to join sequencesencoding Bestrophin to nucleotide sequence encoding a polypeptidedomain, which will facilitate purification of soluble proteins. Suchpurification facilitating domains include, but are not limited to, metalchelating peptides such as histidine-tryptophan modules that allowpurification on immobilised metals, protein A domains that allowpurification on immobilised immunoglobulin, and the domain utilised inthe FLAG extension affinity purification system (Immunex Corp., Seattle,Wash.) The inclusion of cleavable linker sequences such as thosespecific for Factor XA or Enterokinase (Invitrogen, San Diego, Calif.)between the purification domain and Bestrophin may be used to facilitatepurification. One such expression vector provides for expression of afusion protein containing Bestrophin and a nucleic acid encoding 6histidine residues preceding a Thioredoxine or an Enterokinase cleavagesite. The histidine residues facilitate purification on IMIAC(immobilised metal ion affinity chromatography as described in Porath,J. et al. (1992, Prot. Exp. Purif. 3: 263-281)) while the Enterokinasecleavage site provides a means for purifying Bestrophin from the fusionprotein. A discussion of vectors which contain fusion proteins isprovided in Kroll, D. J. et al. (1993; DNA Cell Biol. 12:441-453). Inaddition to recombinant production, fragments of Bestrophin may beproduced by direct peptide synthesis using solid-phase techniques(Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesismay be performed using manual techniques or by automation. Automatedsynthesis may be achieved, for example, using Applied Biosystems 431Apeptide synthesiser (Perkin Elmer). Various fragments of Bestrophin maybe chemically synthesised separately and combined using chemical methodsto produce the full length molecule. The data disclosed in thisinvention show that the nucleic acids and proteins of the invention andeffectors thereof are useful in diagnostic and therapeutic applicationsimplicated, for example but not limited to, in metabolic disorders suchas obesity as well as related disorders such as eating disorder,cachexia, diabetes mellitus, hypertension, coronary heart disease,hypercholesterolemia, osteoarthritis, gallstones, cancers of thereproductive organs, and sleep apnea. Hence, diagnostic and therapeuticuses for the Bestrophin nucleic acids and proteins are, for example butnot limited to, the following: (i) protein therapeutic, (ii) smallmolecule drug target, (iii) antibody target (therapeutic, diagnostic,drug targeting/cytotoxic antibody), (iv) diagnostic and/or prognosticmarker, (v) gene therapy (gene delivery/gene ablation), (vi) researchtools, and (vii) tissue regeneration in vitro and in vivo (regenerationfor all these tissues and cell types composing these tissues and celltypes derived from these tissues).

The nucleic acids and proteins of the invention are particularly usefulin diagnostic and therapeutic applications as described below. Forexample, but not limited to, cDNAs encoding the Bestrophin proteins ofthe invention and particularly their human homologues may be useful ingene therapy, and the Bestrophin proteins of the invention andparticularly their human homologues may be useful when administered to asubject in need thereof. By way of non-limiting example, thecompositions of the present invention will have efficacy for preventionor treatment of patients suffering from diseases and disorders asdescribed above.

Bestrophin nucleic acids, or fragments thereof, may further be useful indiagnostic applications, wherein the presence or amount of the nucleicacids or the proteins are to be assessed. Bestrophin proteins orfragments thereof are further useful in the generation of antibodiesthat bind immunospecifically to the protein of the invention for use intherapeutic or diagnostic methods.

For example, in one aspect, antibodies which are specific for Bestrophinmay be used directly as an antagonist, or indirectly as a targeting ordelivery mechanism for bringing a pharmaceutical agent to cells ortissue which express Bestrophin. The antibodies may be generated usingmethods that are well known in the art. Such antibodies may include, butare not limited to, polyclonal, monoclonal, chimeric, single chain, Fabfragments, and fragments produced by a Fab expression library.Neutralising antibodies, (i.e., those which inhibit dimer formation) areespecially preferred for therapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunised by injectionwith Bestrophin any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminiumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,and dinitrophenol. Among adjuvants used in human, BCG (BacilleCalmette-Guerin) and Corynebacterium parvum are especially preferable.It is preferred that the peptides, fragments, or oligopeptides used toinduce antibodies to Bestrophin have an amino acid sequence consistingof at least five amino acids, and more preferably at least 10 aminoacids. It is preferable that they are identical to a portion of theamino acid sequence of the natural protein, and they may contain theentire amino acid sequence of a small, naturally occurring molecule.Short stretches of Bestrophin amino acids may be fused with those of aheterologous protein such as keyhole limpet hemocyanin and antibodiesproduced against the chimeric molecule.

Monoclonal antibodies to Bestrophin may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Köhler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al. (1984) Mol.Cell Biol. 62:109-120).

In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used. (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceBestrophin-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobulin libraries(Burton, D. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3). Antibodies mayalso be produced by inducing in vivo production in the lymphocytepopulation or by screening recombinant immunoglobulin libraries orpanels of highly specific binding reagents as disclosed in theliterature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

Fragments of anti-Bestrophin antibodies, which contain specific bindingsites for Bestrophin, may also be generated. For example, such fragmentsinclude, but are not limited to, the F(ab′)₂ fragments which can beproduced by Pepsin digestion of the antibody molecule and the Fabfragments which can be generated by reducing the disulfide bridges ofF(ab′)₂ fragments. Alternatively, Fab expression libraries may beconstructed to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity (Huse, W. D. et al. (1989)Science 254:1275-1281).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding and immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between Bestrophins and their specific-antibody.

In another embodiment of the invention, the polynucleotides encodingBestrophin, or any fragment thereof, or effector nucleic acids such asaptamers, antisense molecules, ribozymes or RNAi molecules may be usedfor therapeutic purposes. In one aspect, aptamers, i.e. nucleic acidmolecules capable of binding to a target protein and modulating itsactivity may be obtained by known methods, e.g. by affinity selection ofcombinatorial nucleic acid libraries.

In a further aspect, antisense molecules may be used in situations inwhich it would be desirable to block the transcription of the mRNA. Inparticular, cells may be transformed with sequences complementary topolynucleotides encoding Bestrophin. Thus, antisense molecules may beused to modulate Bestrophin activity, or to achieve regulation of genefunction. Such technology is now well know in the art, and sense orantisense oligomers or larger fragments, can be designed from variouslocations along the coding and/or control regions of sequences encodingBestrophin. Expression vectors derived from retroviruses, adenovirus,herpes or vaccinia viruses, or from various bacterial plasmids may beused for delivery of nucleotide sequences to the targeted organ, tissueor cell population. Methods, which are well known to those skilled inthe art, can be used to construct recombinant vectors, which willexpress antisense molecules complementary to the polynucleotides of thegene encoding Bestrophin. These techniques are described both inSambrook et al. (supra) and in Ausubel et al. (supra). Genes encodingBestrophin can be turned off by transforming a cell or tissue withexpression vectors which express high levels of polynucleotide orfragment thereof which encodes Bestrophin. Such constructs may be usedto introduce untranslatable sense or antisense sequences into a cell.Even in the absence of integration into the DNA, such vectors maycontinue to transcribe RNA molecules until they are disabled byendogenous nucleases. Transient expression may last for a month or morewith a non-replicating vector and even longer if appropriate replicationelements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning antisense molecules, e.g. DNA, RNA, or nucleic acid analoguessuch as PNA, to the control regions of the gene encoding Bestrophin,i.e., the promoters, enhancers, and/or introns. Oligonucleotides derivedfrom the transcription initiation site, e.g., between positions −10 and+10 from the start site, are preferred. Similarly, inhibition can beachieved using “triple helix” base-pairing methodology. Triple helixpairing is useful because it cause inhibition of the ability of thedouble helix to open sufficiently for the binding of polymerases,transcription factors, or regulatory molecules. Recent therapeuticadvances using triplex DNA have been described in the literature (Gee,J. E. et al. (1994) In; Huber, B. E. and B. I. Carr, Molecular andImmunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.). Theantisense molecules may also be designed to block translation of mRNA bypreventing the transcript from binding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyse thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridisation of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage.Examples, which may be used, include engineered hammerhead motifribozyme molecules that can be specifically and efficiently catalyseendonucleolytic cleavage of target sequences. Specific ribozyme cleavagesites within any potential RNA target are initially identified byscanning the target molecule for ribozyme cleavage sites which includethe following sequences: GUA, GUU, and GUC. Once identified, short RNAsequences of between 15 and 20 ribonucleotides corresponding to theregion of the target gene containing the cleavage site may be evaluatedfor secondary structural features which may render the oligonucleotideinoperable. The suitability of candidate targets may also be evaluatedby testing accessibility to hybridisation with complementaryoligonucleotides using ribonuclease protection assays.

Effector nucleic acids such as antisense molecules and ribozymes may beprepared by any method known in the art for the synthesis of nucleicacid molecules. These include techniques for chemically synthesisingoligonucleotides such as solid phase phosphoramidite chemical synthesis.Alternatively, RNA molecules may be generated by in vitro and in vivotranscription of DNA sequences. Such DNA sequences may be incorporatedinto a variety of vectors with suitable RNA polymerase promoters such asT7 or SP6. Alternatively, these cDNA constructs that synthesise RNAconstitutively or inducibly can be introduced into cell lines, cells, ortissues. RNA molecules may be modified to increase intracellularstability and half-life. Possible modifications include, but are notlimited to, the addition of flanking sequences at the 5′ and/or 3′ endsof the molecule or the use of phosphorothioate or 2′ O-methyl ratherthan phosphodiesterase linkages within the backbone of the molecule.This concept is inherent in the production of PNAs and can be extendedin all of these molecules by the inclusion of non-traditional bases suchas inosine, queosine, and wybutosine, as well as acetyl-, methyl-,thio-, and similarly modified forms of adenine, cytidine, guanine,thymine, and uridine which are not as easily recognised by endogenousendonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection and by liposome injections may beachieved using methods, which are well known in the art. Any of thetherapeutic methods described above may be applied to any suitablesubject including, for example, mammals such as dogs, cats, cows,horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical composition, in conjunction with a pharmaceuticallyacceptable carrier, for any of the therapeutic effects discussed above.Such pharmaceutical compositions may consist of Bestrophin, comprise asan active ingredient the protein, the nucleic acid coding therefor or areceptor recognizing the protein or the nucleic acid, e.g. antibodies toBestrophin, mimetics, agonists, antagonists, activators or inhibitors ofBestrophin. The compositions may be administered alone or in combinationwith at least one other agent, such as stabilising compound, which maybe administered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs or hormones. The pharmaceuticalcompositions utilised in this invention may be administered by anynumber of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, transdermal, subcutaneous, intraperitoneal,intranasal, enteral, topical, sublingual, or rectal means.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries, which facilitate processing of the activecompounds into preparations which, can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilising processes. Afterpharmaceutical compositions have been prepared, they can be placed in anappropriate container and labelled for treatment of an indicatedcondition. For administration of Bestrophin, such labelling wouldinclude amount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart. For any compounds, the therapeutically effective does can beestimated initially either in cell culture assays, e.g., of preadipocytecell lines, or in animal models, usually mice, rabbits, dogs, or pigs.The animal model may also be used to determine the appropriateconcentration range and route of administration. Such information canthen be used to determine useful doses and routes for administration inhumans. A therapeutically effective dose refers to that amount of activeingredient, for example Bestrophin, fragments thereof, antibodies ofBestrophin, which is effective against a specific condition. Therapeuticefficacy and toxicity may be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., ED50 (thedose therapeutically effective in 50% of the population) and LD50 (thedose lethal to 50% of the population). The dose ratio betweentherapeutic and toxic effects is the therapeutic index, and it can beexpressed as the ratio, LD50/ED50. Pharmaceutical compositions, whichexhibit large therapeutic indices, are preferred. The data obtained fromcell culture assays and animal studies is used in formulating a range ofdosage for human use. The dosage contained in such compositions ispreferably within a range of circulating concentrations that include theED50 with little or no toxicity. The dosage varies within this rangedepending upon the dosage from employed, sensitivity of the patient, andthe route of administration. The exact dosage will be determined by thepractitioner, in light of factors related to the subject that requirestreatment. Dosage and administration are adjusted to provide sufficientlevels of the active moiety or to maintain the desired effect. Factors,which may be taken into account, include the severity of the diseasestate, general health of the subject, age, weight, and gender of thesubject, diet, time and frequency of administration, drugcombination(s), reaction sensitivities, and tolerance/response totherapy Long-acting pharmaceutical compositions may be administeredevery 3 to 4 days, every week, or once every two weeks depending onhalf-life and clearance rate of the particular formulation. Normaldosage amounts may vary from 0.1 to 100,000 micrograms, up to a totaldose of about 1 g, depending upon the route of administration. Guidanceas to particular dosages and methods of is delivery is provided in theliterature and generally available to practitioners in the art. Thoseskilled in the art employ different formulations for nucleotides thanfor proteins or their inhibitors. Similarly, delivery of polynucleotidesor polypeptides will be specific to particular cells, conditions,locations, etc.

In another embodiment, antibodies may be used for the diagnosis ofconditions or diseases characterised by or associated with over- orunderexpression of Bestrophin, or in assays to monitor patients beingtreated with Bestrophin, agonists, antagonists or inhibitors. Theantibodies useful for diagnostic purposes may be prepared in the samemanner as those described above for therapeutics. Diagnostic assays forBestrophin include methods, which utilise the antibody and a label todetect Bestrophin in human body fluids or extracts of cells or tissues.The antibodies may be used with or without modification, and may belabelled by joining them, either covalently or non-covalently, with areporter molecule. A wide variety of reporter molecules which are knownin the art may be used several of which are described above.

A variety of protocols including ELISA, RIA, and FACS for measuringBestrophin are known in the art and provide a basis for diagnosingaltered or abnormal levels of Bestrophin expression. Normal or standardvalues for Bestrophin expression may be established by combining bodyfluids or cell extracts taken from normal mammalian subjects, preferablyhuman subjects, with antibodies to Bestrophin under conditions suitablefor complex formation. The amount of standard complex formation may bequantified by various methods, but preferably by photometry, means.Quantities of Bestrophin expressed in control and disease samples e.g.from biopsied tissues may be compared with the standard values.Deviation between standard and subject values establishes the parametersfor diagnosing disease.

In another embodiment of the invention, the polynucleotides specific forBestrophin may be used for diagnostic purposes. The polynucleotides,which may be used, include oligonucleotide sequences, antisense RNA andDNA molecules, and nucleic acid analogues such as PNAs. Thepolynucleotides may be used to detect and quantitate gene expression insamples, e.g. in biopsied tissues in which expression is correlated withdisease. The diagnostic assay may be used to distinguish betweenabsence, presence, and excess protein expression, and/or to monitorregulation of Bestrophin levels during therapeutic intervention.

In one aspect, hybridisation with probes and/or primers which arecapable of detecting polynucleotide sequences, including genomicsequences, encoding Bestrophin closely related molecules, may be used toidentify nucleic acid sequences which encode Bestrophin. The specificityof the probe, whether it is made from a highly specific region, e.g.,unique nucleotides in the 5′ regulatory region, or a less specificregion, e.g., especially in the 3′ coding region, and the stringency ofthe hybridisation or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding Bestrophin or alleles, or related sequences. Probesmay also be used for the detection of related sequences, and shouldpreferably contain at least 50% of the nucleotides from any of theBestrophin encoding sequences. The hybridisation probes of the subjectinvention may be DNA, RNA or nucleic acid analogues which are preferablyderived from the nucleotide sequence of human Bestrophin cDNAs or RNAs,or from a genomic sequence including promoter, enhancer elements, and/orintrons of the naturally occurring sequence. Means for producingspecific hybridisation probes for DNAs encoding Bestrophin include thecloning of nucleic acid sequences encoding Bestrophin derivatives intovectors for the production of mRNA probes. Such vectors are known in theart, commercially available, and may be used to synthesise RNA probes invitro by means of the addition of the appropriate RNA polymerases andthe appropriate labelled nucleotides. Hybridisation probes may belabelled by a variety of reporter groups, for example, radionuclidessuch as ³²P or ³⁵S, or enzymatic labels, such as alkaline phosphatasecoupled to the probe via avidin/biotin coupling systems, and the like.

Polynucleotide sequences specific for Bestrophin (for example, VDM2,VDM2-like protein 1, VDM2-like protein 2, or VDM2-like protein 3) may beused for the diagnosis of conditions or diseases, which are associatedwith expression of Bestrophin. Examples of such conditions or diseasesinclude, but are not limited to, metabolic diseases and disorders, suchas obesity and diabetes. Polynucleotide sequences may also be used tomonitor the progress of patients receiving treatment for metabolicdiseases and disorders, including obesity and diabetes. Thepolynucleotide sequences encoding Bestrophin may be used in Southern orNorthern analysis, dot blot, or other membrane-based technologies; inPCR technologies; or in dip stick, pin, ELISA or chip assays utilisingfluids or tissues from patient biopsies to detect altered Bestrophinexpression. Such qualitative or quantitative methods are well known inthe art.

In a particular aspect, the nucleotide sequences may be useful in assaysthat detect activation or induction of various metabolic diseases suchas obesity as well as related disorders such as eating disorder,cachexia, diabetes mellitus, hypertension, coronary heart disease,hyperchotesterolemia, osteoarthritis, gallstones, cancers of thereproductive organs, and sleep apnea. The nucleotide sequences may belabelled by standard methods, and added to a fluid or tissue sample froma patient under conditions suitable for the formation of hybridisationcomplexes. After a suitable incubation period, the sample is washed andthe signal is quantitated and compared with a standard value. Thepresence of signal values corresponding to altered levels of nucleotideBestrophin sequences in the sample indicates the presence of theassociated disease. Such assays may also be used to evaluate theefficacy of a particular therapeutic treatment regimen in animalstudies, in clinical trials, or in monitoring the treatment of anindividual patient.

In order to provide a basis for the diagnosis of disease associated withexpression of Bestrophin, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human subjectswith a sequence, suitable as a probe or a primer, under conditionssuitable for hybridisation or amplification. Standard hybridisation maybe quantified by comparing the values obtained from normal subjects withthose from an experiment where a known amount of a substantiallypurified polynucleotide is used. Standard values obtained from normalsamples may be compared with values obtained from samples from patientswho are symptomatic for disease. Deviation between standard and subjectvalues is used to establish the presence of disease. Once disease isestablished and a treatment protocol is initiated, hybridisation assaysmay be repeated on a regular basis to evaluate whether the level ofexpression in the patient begins to approximate that, which is observedin the normal patient. The results obtained from successive assays maybe used to show the efficacy of treatment over a period ranging fromseveral days to months.

With respect to metabolic diseases such as obesity as well as relateddisorders such as eating disorder, cachexia, diabetes mellitus,hypertension, coronary heart disease, hypercholesterolemia,osteoarthritis, gallstones, cancers of the reproductive organs, andsleep apnea the presence of a relatively high amount of transcript inbiopsied tissue from an individual may indicate a predisposition for thedevelopment of the disease, or may provide a means for detecting thedisease prior to the appearance of actual clinical symptoms. A moredefinitive diagnosis of this type may allow health professionals toemploy preventative measures or aggressive treatment earlier therebypreventing the development or further progression of the pancreaticdiseases and disorders. Additional diagnostic uses for oligonucleotidesmay involve the use of PCR. Such oligomers may be chemicallysynthesised, generated enzymatically, or produced from a recombinantsource. Oligomers will preferably consist of two nucleotide sequences,one with sense orientation (5′.fwdarw.3′) and another with antisense(3′.rarw.5′), employed under optimised conditions for identification ofa specific gene or condition. The same two oligomers, nested sets ofoligomers, or even a degenerate pool of oligomers may be employed underless stringent conditions for detection and/or quantification of closelyrelated DNA or RNA sequences.

Methods which may also be used to quantitate the expression ofBestrophin include radiolabelling or biotinylating nucleotides,coamplification of a control nucleic acid, and standard curves ontowhich the experimental results are interpolated (Melby, P. C. et al.(1993) J. Immunol. Methods, 159:235-244; Duplaa, C. et al. (1993) Anal.Biochem. 212:229-236). The speed of quantification of multiple samplesmay be accelerated by running the assay in an ELISA format where theoligomer of interest is presented in various dilutions and aspectrophotometric or calorimetric response gives rapid quantification.

In another embodiment of the invention, the nucleic acid sequencesspecific for Bestrophin may also be used to generate hybridisationprobes, which are useful for mapping the naturally occurring genomicsequence. The sequences may be mapped to a particular chromosome or to aspecific region of the chromosome using well known techniques. Suchtechniques include FISH, FACS, or artificial chromosome constructions,such as yeast artificial chromosomes, bacterial artificial chromosomes;bacterial P1 constructions or single chromosome cDNA libraries asreviewed in Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J.(1991) Trends Genet. 7:149-154. FISH (as described in Verma et al.(1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press,New York, N.Y.) may be correlated with other physical chromosome mappingtechniques and genetic map data. Examples of genetic map data can befound in the 1994 Genome Issue of Science (265:1981f). Correlationbetween the location of the gene encoding Bestrophin on a physicalchromosomal map and a specific disease, or predisposition to a specificdisease, may help to delimit the region of DNA associated with thatgenetic disease.

The nucleotide sequences of the subject invention may be used to detectdifferences in gene sequences between normal, carrier or affectedindividuals. In situ hybridization of chromosomal preparations andphysical mapping techniques such as linkage analysis using establishedchromosomal markers may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms or parts thereof, by physical mapping. This providesvaluable information to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, for example, AT to 11 q22-23 (Gatti, R. A. etal. (1988) Nature 336:577-580), any sequences mapping to that area mayrepresent associated or regulatory genes for further investigation. Thenucleotide sequences of the subject invention may also be used to detectdifferences in the chromosomal location due to translocation, inversion,etc. among normal, carrier or affected individuals.

In another embodiment of the invention, the proteins of the invention,its catalytic or immunogenic fragments or oligopeptides thereof, an invitro model, a genetically altered cell or animal, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. One can identify effectors, e.g. receptors, enzymes, ligandsor substrates that bind to, modulate or mimic the action of one or moreof the proteins of the invention. The protein or fragment thereofemployed in such screening may be free in solution, affixed to a solidsupport, borne on a cell surface, or located intracellularly. Theformation of binding complexes, between the proteins of the inventionand the agent tested, may be measured. Agents could also, eitherdirectly or indirectly, influence the activity of the proteins of theinvention. Target mechanisms could for example include the chloridechannel or other conductance activities of bestrophin as well as theregulation of bestrophin activity by phosphorylation anddephosphorylation or other posttranslational modifications. Moreover,agents could interfere with the dimerization or oligomerization ofbestrophins or, in a heterologous manner, of bestrophins with otherproteins, e.g. ion channels. Of particular interest are screening assaysfor agents that have a low toxicity for mammalian cells. The term“agent” as used herein describes any molecule, e.g. protein orpharmaceutical, with the capability of altering or mimicking thephysiological function of one or more of the proteins of the invention.Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 50 and less than about 2,500 Daltons.Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise carbocyclic or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups.

Candidate agents are also found among biomolecules including peptides,saccharides, fatty acids, steroids, purines, pyrimidines, nucleic acidsand derivatives, structural analogs or combinations thereof. Candidateagents are obtained from a wide variety of sources including librariesof synthetic or natural compounds. For example, numerous means areavailable for random and directed synthesis of a wide variety of organiccompounds and biomolecules, including expression of randomizedoligonucleotides and oligopeptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs. Where the screening assay is a binding assay, one ormore of the molecules may be joined to a label, where the label candirectly or indirectly provide a detectable signal.

Another technique for drug screening, which may be used, provides forhigh throughput screening of compounds having suitable binding affinityto the protein of interest as described in published PCT applicationWO84/03564. In this method, as applied to the proteins of the inventionlarge numbers of different small test compounds, e.g. aptamers,peptides, low-molecular weight compounds etc., are provided orsynthesized on a solid substrate, such as plastic pins or some othersurface. The test compounds are reacted with the proteins or fragmentsthereof, and washed. Bound proteins are then detected by methods wellknown in the art. Purified proteins can also be coated directly ontoplates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support. In another embodiment, onemay use competitive drug screening assays in which neutralizingantibodies capable of binding the protein specifically compete with atest compound for binding the protein. In this manner, the antibodiescan be used to detect the presence of any peptide, which shares one ormore antigenic determinants with the protein.

The nucleic acids encoding the proteins of the invention can be used togenerate transgenic cell lines and animals. These transgenic animals areuseful in the study of the function and regulation of the proteins ofthe invention in vivo. Transgenic animals, particularly mammaliantransgenic animals, can serve as a model system for the investigation ofmany developmental and cellular processes common to humans. Transgenicanimals may be made through homologous recombination in embryonic stemcells, where the normal locus of the gene encoding the protein of theinvention is mutated. Alternatively, a nucleic acid construct encodingthe protein is injected into oocytes and is randomly integrated into thegenome. One may also express the genes of the invention or variantsthereof in tissues where they are not normally expressed or at abnormaltimes of development. Furthermore, variants of the genes of theinvention like specific constructs expressing anti-sense molecules orexpression of dominant negative mutations, which will block or alter theexpression of the proteins of the invention may be randomly integratedinto the genome. A detectable marker, such as lac Z or luciferase may beintroduced into the locus of the genes of the invention, whereupregulation of expression of the genes of the invention will result inan easily detectable change in phenotype. Vectors for stable integrationinclude plasmids, retroviruses and other animal viruses, yeastartificial chromosomes (YACs), and the like. DNA constructs forhomologous recombination will contain at least portions of the genes ofthe invention with the desired genetic modification, and will includeregions of homology to the target locus. Conveniently, markers forpositive and negative selection are included. DNA constructs for randomintegration do not need to contain regions of homology to mediaterecombination. DNA constructs for random integration will consist of thenucleic acids encoding the proteins of the invention, a regulatoryelement (promoter), an intron and a poly-adenylation signal. Methods forgenerating cells having targeted gene modifications through homologousrecombination are known in the field. For embryonic stem (ES) cells, anES cell line may be employed, or embryonic cells may be obtained freshlyfrom a host, e.g. mouse, rat, guinea pig, etc. Such cells are grown onan appropriate fibroblast-feeder layer and are grown in the presence ofleukemia inhibiting factor (LIF). ES or embryonic cells may betransfected and can then be used to produce transgenic animals. Aftertransfection, the ES cells are plated onto a feeder layer in anappropriate medium. Cells containing the construct may be selected byemploying a selection medium. After sufficient time for colonies togrow, they are picked and analyzed for the occurrence of homologousrecombination. Colonies that are positive may then be used for embryomanipulation and morula aggregation. Briefly, morulae are obtained from4 to 6 week old superovulated females, the Zona Pellucida is removed andthe morulae are put into small depressions of a tissue culture dish. TheES cells are trypsinized, and the modified cells are placed into thedepression closely to the morulae. On the following day the aggregatesare transfered into the uterine horns of pseudopregnant females. Femalesare then allowed to go to term. Chimeric offsprings can be readilydetected by a change in coat color and are subsequently screened for thetransmission of the mutation into the next generation (F1-generation).Offspring of the F1-generation are screened for the presence of themodified gene and males and females having the modification are mated toproduce homozygous progeny. If the gene alterations cause lethality atsome point in development, tissues or organs can be maintained asallogenic or congenic grafts or transplants, or in vitro culture. Thetransgenic animals may be any non-human mammal, such as laboratoryanimal, domestic animals, etc., for example, mouse, rat, guinea pig,sheep, cow, pig, and others. The transgenic animals may be used infunctional studies, drug screening, and other applications and areuseful in the study of the function and regulation of the proteins ofthe invention in vivo.

Finally, the invention also relates to a kit comprising at least one of

-   (a) a Bestrophin nucleic acid molecule or a fragment thereof;-   (b) a vector comprising the nucleic acid of (a);-   (c) a host cell comprising the nucleic acid of (a) or the vector of    (b);-   (d) a polypeptide encoded by the nucleic acid of (a);-   (e) a fusion polypeptide encoded by the nucleic acid of (a);-   (f) an antibody, an aptamer or another receptor against the nucleic    acid of (a) or the polypeptide of (d) or (e) and-   (g) an anti-sense oligonucleotide of the nucleic acid of (a).

The kit may be used for diagnostic or therapeutic purposes or forscreening applications as described above. The kit may further containuser instructions.

The Figures show:

FIG. 1 shows the increase of triglyceride content of EP(3)32517 andHD-EP(3)36237 flies caused by homozygous viable integration of theP-vector (in comparison to controls).

FIG. 2 shows the molecular organisation of the mutated Bestrophin(Best1, CG6264) gene locus.

FIG. 3 shows the comparison (CLUSTAL X 1.8) of human Bestrophin proteinswith the homologue Drosophila Bestrophin protein. Gaps in the alignmentare represented as -. In the Figure, chr 12′ refers to human VDM2-likeprotein 3, chr1GENSCAN predicted_peptide′ refers to human VDM2-likeprotein 2, hsXP_(—)043405′ refers to human VDM2 protein, hs NP_(—)060152mod′ refers to a protein similar to human VDM2-like protein 1, anddmbest′ refers to Drosophila melanogaster bestrophin.

FIG. 4 shows the nucleotide sequence of four human Bestrophin homologues(SEQ ID NO. 1-8).

FIG. 5 shows the comparison (CLUSTAL W 1.82 multiple sequence alignment)of human, mouse, and Drosophila Bestrophin proteins. Gaps in thealignment are represented as—

-   Bestrph_Hs refers to |SWISS-PROT Accession Number 076090 or GenBank    Accession Number NP_(—)004174;-   Bestrph_Mm refers to Mouse EnsEMBL Genscan predicted peptide    19.9000001-10000000.20.329852.331536;-   VMDY2_(—)3_Hs GenBank Accession Number AF440758_(—)1, human    vitelliform macular dystrophy 2-like protein 3;-   VMDY2_3_Mm refers to ENSEMBL Accession Number ENSMUSP00000020378;-   VMDY2_(—)1_Hs refers to GenBank Accession Number AF440756_(—)1,    human vitelliform macular dystrophy 2-like protein 1;-   VMDY2_(—)1 Mm refers to GenBank Accession Number NP_(—)663363 or    ENSEMBL GenBank Accession Number ENSMUSP00000005276;-   VMDY2_(—)2 Hs refers to GenBank Accession Number AF440757_(—)1,    human vitelliform macular dystrophy 2-like protein 2 [Homo sapiens];-   VMDY2_(—)2 Mm refers to ENSEMBL Accession Number ENSMUSP00000049289;    and Bestrph_Dm′ refers to GenBank Accession Number NP_(—)652603.1.

FIG. 6 shows the analysis of vitelliform macular dystrophy 2-likeprotein 3 expression in mammalian tissues.

FIG. 6A. Real time PCR of VDM2-like protein 3 mRNA expression in mousewildtype tissues.

FIG. 6B. VDM2-like protein 3 mRNA expression in mice-expressing leptin(wt mice) compared to genetically obese (ob/ob) mice without leptinexpression and to fasted mice (fasted-mice).

FIG. 6C. Real-time PCR mediated analysis of vitelliform maculardystrophy 2 (VDM2)-like protein 3 mRNA in genetically obese (db/db) micewithout leptin receptor expression compared to wild-type (wt) mice.

FIG. 6D. Expression of VDM2-like protein 3 mRNA in high fat (palmitat)diet-mice compared to mice fed a normal diet.

FIG. 6E. Expression of VDM-like protein 3 mRNA in mammalian fibroblast(3T3-L1) cells, during the differentiation from pre-adipocytes to matureadipocytes.

FIG. 7A shows the expression of mouse vitelliform macular dystrophyprotein (VDM2) mRNA in different mouse tissues.

FIG. 7B shows the expression vitelliform macular dystrophy (VDM2)protein mRNA in different mouse models, (wildtype mice, wt; geneticallyobese mice, ob/ob fasted mice).

FIG. 7C shows the expression of VDM2 in mice under a high fat (palmitat)diet compared to mice fed a normal diet.

FIG. 8 shows the expression profiling of mouse vitelliform maculardystrophy 2-like protein 1 mRNA.

FIG. 9 shows the expression of VMD2 mRNA in different human tissues.

FIG. 10 shows the expression of VMD2-like protein 3 mRNA in differenthuman tissues.

THE EXAMPLES ILLUSTRATE THE INVENTION Example 1 Measurement ofTriglyceride Content of Mutated Flies

The average increase of triglyceride content of homozygous HD-EP(3)32517and HD-EP(3)36237 flies was investigated in comparison to control flies(FIG. 1). For determination of triglyceride, flies were incubated for 5min at 90° C. in an aqueous buffer using a waterbath, followed by hotextraction. After another 5 min incubation at 90° C. and mildcentrifugation, the triglyceride content of the flies extract wasdetermined using Sigma Triglyceride (INT 336-10 or -20) assay bymeasuring changes in the optical density according to the manufacturer'sprotocol. As a reference protein content of the same extract wasmeasured using BIO-RAD DC Protein Assay according to the manufacturer'sprotocol. The assay was repeated several times.

The average triglyceride level of EP collection is shown as 100% inFIG. 1. HD-EP(3)32517 homozygous flies show constantly a highertriglyceride content than the controls (260%). In addition HD-EP(3)36237homozygous flies also show constantly a higher triglyceride content thanthe controls (160%). Therefore, the loss of gene activity in the locus85F13-85F14 (estimated), where the EP-vector of HD-EP(3)32517 andHD-EP(3)36237 flies is homozygous viably integrated, is responsible forchanges in the metabolism of the energy storage triglycerides, thereforerepresenting in both cases an obese fly model.

Example 2 Identification of the Bestrophin Gene

In FIG. 2, genomic DNA sequence is represented by the assembly as adotted black line (from position 5985465 to 5997965 on chromosome 3R)that includes the integration sites of HD-EP(3)32517 and HD-EP(3)36237.Transcribed DNA sequences (ESTs) and predicted exons are shown as barsin the lower two lines. Predicted exons of gene CG6264 encodingBestrophin (Best1; GadFly release 3, Bestrophin 1) are shown as darkgrey bars and introns as light grey bars. Bestrophin encodes for a genethat is predicted by GadFly sequence analysis programs as CG6264′Genbank Accession Number NP_(—)652603.1). Using plasmid rescue methodgenomic DNA sequences that are directly localised 5′ or 3′ of theHD-EP(3)32517 and HD-EP(3)36237 integration site were isolated. Publicsequence databases were screened ‘thereby ’ identifying the integrationsite of HD-EP(3)32517 and HD-EP(3)36237 causing an increase oftriglyceride content. HD-EP(3)32517 is integrated in the 5′ exon of thegene Best1 (CG6264) and HD-EP(3)36237 is integrated into theenhancer/promoter region 5′ of Best1 (CG6264). Therefore, expression ofthe gene Best1 (CG6264) could be effected by homozygous viableintegration of HD-EP(3)32517 and HD-EP(3)36237 leading to increase ofthe energy storage triglycerides.

Example 3 Identification of Human Bestrophin Homologues

Bestrophin homologous proteins and nucleic acid molecules codingtherefore are obtainable from insect or vertebrate species, e.g. mammalsor birds. Particularly preferred are human Bestrophin homologous nucleicacids and polypeptides encoded thereby, particularly encoding (i) ahuman Bestrophin protein on chromosome 11 (or also refered to as humanvitelliform macular dystrophy, VMD2; Genbank Accession No. NP_(—)004174;Swiss Prot. Accession Number 076090; formerly Genbank Accession No.XM_(—)043405, see FIGS. 4A and 4B; SEQ ID NO. 1/2), or (ii) a Bestrophinhomologous protein on human chromosome 12 (genomic sequence GenbankAccession No. AC016153, see FIGS. 4E and 4F; SEQ ID NO. 5/6); identicalto human vitelliform macular dystrophy 2-like protein 3; GenbankAccession Number AF440758_(—)1, (iii) a Bestrophin homologous protein onhuman chromosome 1 (genomic sequence Genbank Accession No. AL592166, seeFIGS. 4G and 4H; SEQ ID NO. 7/8) identical to encoding a humanvitelliform macular dystrophy 2-like protein 2; Genbank Accession NumberAF440757_(—)1) or (iv) encoding a Bestrophin homologous protein on humanchromosome 19 (assembled from cDNA Genbank Accession No. NM_(—)017682and genomic sequence AC018761, see FIGS. 4C and 4D or 4I and 4J; SEQ IDNO. 3/4; similar to human vitelliform macular dystrophy 2-like protein 1(Genbank Accession Number AF440756_(—)1) which has an additional 43amino acids at the amino terminus). An alignment of Bestrophin fromdifferent species has been done by the ClustaW program and isillustrated in FIG. 3 and FIG. 5.

Example 4 Expression of the Polypeptides in Mammalian Tissues

For analyzing the expression of the polypeptides disclosed in thisinvention in mammalian tissues, several mouse strains (preferrably micestrains C57BI/6J, C57BI/6 ob/ob and C57BI/KS db/db which are standardmodel systems in obesity and diabetes research) were purchased fromHarlan Winkelmann (33178 Borchen, Germany) and maintained under constanttemperature (preferrably 22° C.), 40 percent humidity and a light/darkcycle of preferrably 14/10 hours. The mice were fed a standard chow (forexample, from ssniff Spezialitäten GmbH, order number ssniff M-ZV1126-000). Animals were sacrificed at an age of 6 to 8 weeks. Theanimal tissues were isolated according to standard procedures known tothose skilled in the art, snap frozen in liquid nitrogen and stored at−80° C. until needed.

For analyzing the role of the proteins disclosed in this invention inthe in vitro differentiation of different mammalian cell culture cellsfor the conversion of pre-adipocytes to adipocytes, mammalian fibroblast(3T3-L1) cells (e.g., Green & Kehinde, Cell 1: 113-116, 1974) wereobtained from the American Tissue Culture Collection (ATCC, Hanassas,Va., USA; ATCC-CL 173). 3T3-L1 cells were maintained as fibroblasts anddifferentiated into adipocytes as described in the prior art (e.g., Qiu.et al., J. Biol. Chem. 276:11988-95, 2001; Slieker et al., BBRC 251:225-9, 1998). At various time points of the differentiation procedure,beginning with day 0 (day of confluence) and day 2 (hormone addition,for example, dexamethason and 3-isobutyl-1-methylxanthin), up to 10 daysof differentiation, suitable aliquots of cells were taken every twodays.

RNA was isolated from mouse tissues or cell culture cells using TrizolReagent (for example, from Invitrogen, Karlsruhe, Germany) and furtherpurified with the RNeasy Kit (for example, from Qiagen, Germany) incombination with an DNase-treatment according to the instructions of themanufacturers and as known to those skilled in the art. Total RNA wasreverse transcribed (preferrably using Superscript II RNaseH− ReverseTranscriptase, from Invitrogen, Karlsruhe, Germany) and subjected toTaqman analysis preferrably using the Taqman 2×PCR Master Mix (fromApplied Biosystems, Weiterstadt, Germany; the Mix contains according tothe Manufacturer for example AmpliTaq Gold DNA Polymerase, AmpErase UNG,dNTPs with dUTP, passive reference Rox and optimized buffer components)on a GeneAmp 5700 Sequence Detection System (from Applied Biosystems,Weiterstadt, Germany).

Taqman analysis was performed preferrably using the followingprimer/probe pairs:

For the amplification of mouse vitelliform macular dystrophy protein(VMD; mouse EnsEMBL Genscan predicted pep tide19.9000001-10000000.20.329852.331536): Mouse VMD protein forward primer(SEQ ID NO: 9): 5′-AAG GCC TAT CTT GGA GG TCG A-3′; mouse VMD proteinreverse primer (SEQ ID NO: 10): 5′-GTA CAC ACC TCA TTC ATC AGG CTC-3′;Taqman probe (SEQ ID NO: 11): (5/6-FAM) TCC GGG ACA CCG TCC TGC TCC(5/6-TAMRA)

For the amplification of mouse vitelliform macular dystrophy 2-likeprotein 1 (VMD2L1; GenBank Accession Number NP_(—)663363 or ENSEMBLGenBank Accession Number ENSMUSP00000005276): Mouse VMD2L1 forwardprimer (SEQ ID NO: 12): 5′-GCG CTA CGC AGG GCT CT-3′; mouse VMD2L1reverse primer (SEQ ID NO: 13): 5′-CCG TTT GAA GAC TGC TGT GC-3′; Taqmanprobe (SEQ ID NO: 14): (5/6-FAM) CGC GGT GCT GAT CCT TCG TTC TG (5/6-TAMRA)

For the amplification of vitelliform macular dystrophy 2-like protein 3(VMD2L3; ENSEMBL Accession Number ENSMUSP00000020378): Mouse VMD2L3forward primer (SEQ ID NO: 15): 5′-AGG TGG AGC CTG CCA GAG T-3′; mouseVMD2L3 reverse primer (SEQ ID NO: 16): 5′-AGG ATC TGG ACC TAA GTT TCCC-3′; Taqman probe (SEQ ID NO: 17): (5/6-FAM) TCT GGA GTC CAG GCA CACCTC GC (5/6- TAMRA).

As shown in FIG. 6A, real time PCR (Taqman) analysis of the expressionof the VMD2 like protein 3 mRNA in mammalian (mouse) tissues revealedthat VMD2 like protein 3 mRNA is expressed more ubiquitously indifferent mammalian tissues with clear expression in white adiposetissue (WAT) and brown adipose tissue (BAT). In addition, there arehigher levels of expression in muscle, testis, heart, lung, kidney,hypothalamus, and brain tissues. Thus, our analysis shows clearly thatthe expression of VMD2 like protein 3 mRNA is under metabolic control.In genetically obese (ob/ob) mice (mice without leptin expression), theexpression level of VMD2 like protein 3 mRNA is prominentlydown-regulated (85%) in WAT compared to wildtype levels (FIG. 6B). Inaddition, the down-regulation in the VMD2 like protein 3 mRNA expressionin the WAT of ob/ob mice is also seen in the genetically obese mousemodel db/db where the leptin-receptor is missing (see FIG. 6C).Expression of VMD2 like protein 3 mRNA is strongly induced (24 foldupregulation) in the liver of fasted mice (FIG. 6B). In high fat(palmitat) diet mice, the level of VMD2 like protein 3 mRNA issignificantly (86%) reduced in WAT compared to mice fed a standard diet(FIG. 6D). During the differentiation of mammalian fibroblast cells (forexample, 3T3-L1) from pre-adipocytes to mature adipocytes, theexpression of VDM2 like protein 3 is significantly (81%) downregulated(FIG. 6E). This could indicate that VDM2-like protein 3 is an inhibitorof adipocyte lipid accumulation.

As shown in FIG. 7A, real time PCR (Taqman) analysis of the expressionof the VMD2 protein mRNA in mammalian (mouse) tissues revealed that VMD2mRNA is expressed ubiquitously in different mammalian tissues, withclear expression in adipose tissues (WAT and BAT) (FIG. 7A). Our resultsclearly show that the expression of VDM2 mRNA is under metaboliccontrol. In genetically obese (ob/ob) mice, expression of VDM2 mRNA isstrongly induced in WAT (FIG. 7B). In mice under a high fat diet, astrong upregulation of VDM2 mRNA can be observed in muscle (11 fold) andin BAT, see FIG. 7C.

As shown in FIG. 8, real time PCR (Taqman) analysis of the expression ofthe VMD2 like protein 1 protein mRNA in mammalian (mouse) tissuesrevealed that VMD2 like protein 1 mRNA is expressed in differentmammalian tissues, showing highest level of expression in colon andhigher levels in hypothalamus, brain and testis.

No results are shown for VDM2 like protein 2.

Although all proteins are expressed in the hypothalamus and to a lesserdegree in other brain areas they show a clear tissue specific expressionsuggesting distinct roles in the different metabolic requirements ofvarious tissues.

Example 5 Expression of the Polypeptides in Mammalian (Human) Tissues

For analyzing the expression of the polypeptides disclosed in thisinvention in mammalian tissues, human RNAs isolated from differenttissues were obtained from Invitrogen Corp., Karlsruhe, Germany: (i)total RNA from human adult skeletal muscle (Invitrogen Corp. OrderNumber 735030); (ii) total RNA from human adult lung (Invitrogen Corp.Order Number 735020); (iii) total RNA from human adult liver (InvitrogenCorp. Order Number 735018); (iv) total RNA from human adult placenta(Invitrogen Corp. Order Number 735026); (v) total RNA from human adulttestis (Invitrogen Corp. Order Number 64101-1); (vi) total RNA fromhuman normal adipose tissue (Invitrogen Corp. Order Number D6005-01);(vii) total RNA from human normal pancreas (Invitrogen Corp. OrderNumber DG6101); (viii) total RNA from human normal brain (Invitrogen.Corp. Order Number D6030-01).

The RNA was treated with DNase according to the instructions of themanufacturers (for example, from Qiagen, Germany) and as known to thoseskilled in the art. Total RNA was reverse transcribed (preferrably usingSuperscript II RNaseH− Reverse Transcriptase, from Invitrogen,Karlsruhe, Germany) and subjected to Taqman analysis preferrably usingthe Taqman 2×PCR Master Mix′ (from Applied Biosystems, Weiterstadt,Germany). The Taqman 2×PCR Master Mix contains according to theManufacturer for example AmpliTaq Gold DNA Polymerase, AmpErase UNG,dNTPs with dUTP, passive reference Rox and optimized buffer components)on a GeneAmp 5700 Sequence Detection System (all obtained from AppliedBiosystems, Weiterstadt, Germany).

Taqman analysis was performed preferrably using the followingprimer/probe pairs:

For the amplification of VMD2: human VMD2 forward primer (SEQ ID NO:18): 5′-TCA CGC TGG CAT CAT TGG A-3′; human VMD2 reverse primer (SEQ IDNO: 19): 5′-CCC TGG GAG GAT GG TGA TC-3′; Taqman probe (SEQ ID NO: 20):(5/6-FAM) CGC TTC CTA GGC CTG CAG TCC CA (5/6- TAMRA)

For the amplification of VMD2 like protein 3: human VMD2 like3 forwardprimer (SEQ ID NO: 21): 5′-CCC ACC ATA CAC ATT GGC AG-3′; human VMD2like3 reverse primer (SEQ ID NO: 22): 5′-TTT CCC CAT CTG GAC TGT TGA-3′;Taqman probe (SEQ ID NO: 23): (5/6-FAM) TGC TGA CTA CTG CAT ACC CTC ATTTCT GGG T (5/6-TAMRA)

For the amplification of VMD2 like protein 1: human VMD2 like1 forwardprimer (SEQ ID NO: 24): 5′-AGG TCC CTG CAC GGC A-3′; human VMD2 like1reverse primer (SEQ ID NO: 25): 5′-TTT ACA AAG GCA CAC GAG GCT-3′;Taqman probe (SEQ ID NO: 26): (5/6-FAM) CCA CGC AGG TGT CCC GGT CTG(5/6-TAMRA)

For the amplification of VMD2 like protein 2: human VMD2 like2 forwardprimer (SEQ ID NO: 27): 5′-CAT CAC GGA AGG TCT TGT CAA A-3′; human VMD2like2 reverse primer (SEQ ID NO: 28): 5′-TCC CAA ATC TAA CGT GCC AGA-3′;Taqman probe (SEQ ID NO: 29): (5/6-FAM) TGC TGG GCA CCA CTC CCA GCA T(5/6- TAMRA)

As shown in FIG. 9, real time PCR (Taqman) analysis of the expression ofVDM 2 protein in human tissues revealed that VDM2 is expressed indifferent mammalian tissues, showing higher levels of expression inbrain, testis, lung and adipose tissue, suggesting a role in theregulation of energy homeostasis. The data obtained with human tissueare in good agreement with the data obtained with mouse tissues (seeFIG. 7A). Particularly, an upregulation of VMD2 in human preadipocyteswas found.

As shown in FIG. 10, real time PCR (Taqman) analysis of the expressionof VDM2 like protein 3 in human tissues revealed that VDM2 like protein3 is expressed in different human tissues, showing highest level ofexpression in muscle and to a lesser extend in brain and testis. Similarresults were obtained with mouse tissues (see FIG. 6A).

Not shown are the results for VDM2 like protein 1 and VDM2 like protein2 as these genes showed no detectable expression in the tissues analyzedso far.

1. A pharmaceutical composition comprising a nucleic acid molecule ofthe Bestrophin gene family or a polypeptide encoded thereby or afragment or a variant of said nucleic acid molecule or said polypeptideor an antibody, an aptamer or another receptor recognizing a nucleicacid molecule of the Bestrophin gene family or a polypeptide encodedthereby together with pharmaceutically acceptable carriers, diluentsand/or adjuvant.
 2. The composition of claim 1, wherein the nucleic acidmolecule is a vertebrate or insect Bestrophin nucleic acid, particularlya human Bestrophin nucleic acid (i) encoding a Bestrophin homologousprotein 15 on human chromosome 12 (SEQ NO. 5/6); referred to as humanvitelliform macular dystrophy 2-like protein 3, (ii) encoding a humanBestrophin protein on human chromosome 11 (SEQ 10 NO.1/2); referred toas human vitelliform macular dystrophy, VMD2, (iii) encoding aBestrophin homologous protein on human chromosome 1 (SEQ 10 NO. 7/8);referred to as human vitelliform macular dystrophy 2-like protein 2 or(iv) encoding a Bestrophin homologous protein on human chromosome 19(SEQ 10 NO. 3/4); referred to a (human vitelliform macular dystrophy2-like protein 1 or a fragment thereof or a variant thereof.
 3. Thecomposition of claim 1 or 2, wherein said nucleic acid moleculecomprises (a) a Bestrophin nucleotide sequence as shown in FIG. 4 or thecomplementary strand thereof, (b) a nucleotide sequence which hybridizesunder stringent conditions to a nucleic acid molecule encoding aBestrophin amino acid sequence as shown in FIG. 4 or the complementarystrand thereof, (c) a nucleotide sequence corresponding to the sequencesof (a) or (b) within the degeneration of the genetic code, (d) anucleotide sequence which encodes a polypeptide which is at least 85%,preferably at least 90%, more preferably at least 95%, more preferablyat least 98% and up to 99.6% identical to the amino acid sequences shownin FIG. 4, (e) a nucleotide sequence which differs from the nucleic,acid molecule of (a) to (d) by mutation and wherein said mutation causesan alteration, deletion, duplication or premature stop in the encodedpolypeptide or (f) a partial nucleotide sequence of any of the sequencesof (a) to (e) having a length of at least 15 bases, preferably at least20 bases, more preferably at least 25 bases and most preferably at least50 bases.
 4. The composition of claim 1, wherein the nucleic acidmolecule is a DNA molecule, particularly a cDNA or a genomic DNA.
 5. Thecomposition of claim 1, wherein said nucleic acid encodes a polypeptidecontributing to regulating the energy homeostasis and/or the metabolismof triglycerides.
 6. The composition of claim 1, wherein said nucleicacid molecule is a recombinant nucleic acid molecule.
 7. The compositionof claim 1, wherein the nucleic acid molecule is a vector, particularlyan expression vector.
 8. The composition of claim 1, wherein thepolypeptide is a recombinant polypeptide.
 9. The composition of claim 8,wherein said recombinant polypeptide is a fusion polypeptide.
 10. Thecomposition of claim 1, wherein said nucleic acid molecule is selectedfrom hybridization probes, primers and anti-sense oligonucleotides. 11.The composition of claim 1 which is a diagnostic composition.
 12. Thecomposition of claim 1 which is a therapeutic composition.
 13. Thecomposition of claim 1 for the manufacture of an agent for detectingand/or verifying, for the treatment, alleviation and/or prevention of andisorders, including metabolic diseases such as obesity and otherbody-weight regulation disorders as well as related disorders such aseating disorder, cachexia, diabetes mellitus, hypertension, coronaryheart disease, hypercholesterolemia, osteoarthritis, gallstones, cancersof the reproductive organs, and sleep apnea and others, in cells, cellmasses, organs and/or subjects.
 14. Use of a nucleic acid molecule ofthe Bestrophin gene family or a polynucleotide encoded thereby or afragment or a variant of said nucleic acid molecule or said polypeptideor an antibody, an aptamer or another receptor recognizing a nucleicacid molecule of the Bestrophin gene family or a polypeptide encodedthereby for controlling the function of a gene and/or a gene productwhich is influenced and/or modified by a Bestrophin homologouspolypeptide.
 15. Use of the nucleic acid molecule of the Bestrophin genefamily or a polynucleotide encoded thereby or a fragment or a variant ofsaid nucleic acid molecule or said polypeptide or an antibody, anaptamer or another receptor recognizing a nucleic acid molecule of theBestrophin gene family or a polypeptide encoded thereby for identifyingsubstances capable of interacting with a Bestrophin homologouspolypeptide.
 16. A non-human transgenic animal exhibiting a modifiedexpression of a Bestrophin homologous polypeptide.
 17. The animal ofclaim 16, wherein the expression of the Bestrophin homologouspolypeptide is increased and/or reduced.
 18. A recombinant host cellexhibiting a modified expression of an Bestrophin homologouspolypeptide.
 19. The cell of claim 18 which is a human cell.
 20. Amethod of identifying a (poly)peptide involved in the regulation ofenergy homeostasis and/or metabolism of triglycerides in a mammalcomprising the steps of (a) contacting .a collection of (poly)peptideswith a Bestrophin homologous polypeptide or a fragment thereof underconditions that allow binding of said (poly) peptides; (b) removing(poly)peptides which do not bind and (c) identifying (poly)peptides thatbind to said Bestrophin homologous polypeptide.
 21. A method ofscreening for an agent which modulates the interaction of a Bestrophinhomologous polypeptide with a binding target/agent, comprising the stepsof (a) incubating a mixture comprising (aa) a Bestrophin homologouspolypeptide, or a fragment thereof; (ab) a binding target/agent of saidBestrophin homologous polypeptide or fragment thereof; and (ac) acandidate agent. under conditions whereby said Bestrophin polypeptide orfragment thereof specifically binds to said binding target/agent at areference affinity; (b) detecting the binding affinity of saidBestrophin polypeptide or fragment thereof to said binding target todetermine an (candidate) agent-biased affinity; and (c) determining adifference between (candidate) agent-biased affinity and the referenceaffinity.
 22. A method of screening for an agent which modulates theactivity of a Bestrophin homologous polypeptide, comprising the steps:(a) incubating a mixture comprising (aa) a Bestrophin homologouspolypeptide, or a fragment thereof; (ab) a candidate agent; underconditions whereby an activity of said Bestrophin polypeptide orfragment thereof may be determined; (b) determining the activity of saidBestrophin polypeptide or fragment thereof in the presence of saidcandidate agent; and (c) determining a difference between the activityin presence of said candidate agent and a reference activity.
 23. Themethod of claim 22 wherein the activity is selected from a chloridechannel or another conductance activity.
 24. The method of claim 22wherein the activity is selected from the degree of phosphorylation orother posttranslational modifications.
 25. A pharmaceutical compositioncomprising the (poly)peptide identified by the method of claim 20 with apharmaceutically acceptable carrier, diluent and/or adjuvant.
 26. Thecomposition of claim 25 wherein said composition is a pharmaceuticalcomposition for preventing, alleviating or treating of diseases anddisorders, including metabolic diseases such as obesity and otherbody-weight regulation disorders as well as related disorders such aseating disorder, cachexia, diabetes mellitus, hypertension, coronaryheart disease, hypercholesterolemia, osteoarthritis, gallstones, cancersof the reproductive organs, and sleep apnea and other diseases anddisorders.
 27. Use of a (poly)peptide as identified by the method ofclaim 20 for the preparation of a pharmaceutical composition for thetreatment, alleviation and/or prevention of diseases and disorders,including metabolic diseases such as obesity and other body-weightregulation disorders as well as related disorders such as eatingdisorder, cachexia, diabetes mellitus, hypertension, coronary heartdisease, hypercholesterolemia, osteoarthritis, gallstones, cancers ofthe reproductive organs, and sleep apnea and other diseases anddisorders.
 28. Use of a nucleic acid molecule of the Bestrophin familyor of a fragment thereof for the preparation of a non-human animal whichover- or underexpresses the Bestrophin gene product.
 29. Kit comprisingat least one of (a) an Bestrophin nucleic acid molecule or a fragmentthereof; (b) a vector comprising the nucleic acid of (a); (c) a hostcell comprising the nucleic acid of (a) or the vector of (b); (d) apolypeptide encoded by the nucleic acid of (a); (e) a fusion polypeptideencoded by the nucleic acid of (a); (f) an antibody, an aptamer oranother receptor against the nucleic acid of (a) or the polypeptide of(d) or (e) and (g) an anti-sense oligonucleotide of the nucleic acid of(a).
 30. A pharmaceutical composition comprising the agent identified bythe method of claim 21 with a pharmaceutically acceptable carrier,diluent and/or adjuvant.
 31. Use of an agent as identified by the methodof claim 21 for the preparation of a pharmaceutical composition for thetreatment, alleviation and/or prevention of diseases and disorders,including metabolic diseases such as obesity and other body-weightregulation disorders as well as related disorders such as eatingdisorder, cachexia, diabetes mellitus, hypertension, coronary heartdisease, hypercholesterolemia, osteoarthritis, gallstones, cancers ofthe reproductive organs, and sleep apnea and other diseases anddisorders.